fmicb-07-01927 November 25, 2016 Time: 18:18 # 1 ORIGINAL RESEARCH published: 29 November 2016 doi: 10.3389/fmicb.2016.01927 An Integrative Genomic Island Affects the Adaptations of the Piezophilic Hyperthermophilic Archaeon Pyrococcus yayanosii to High Temperature and High Hydrostatic Pressure Zhen Li1,2, Xuegong Li2,3, Xiang Xiao1,2 and Jun Xu1,2* 1 State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China, 2 Institute of Oceanology, Shanghai Jiao Tong University, Shanghai, China, 3 Deep-Sea Cellular Microbiology, Department of Deep-Sea Science, Sanya Institute of Deep-Sea Science and Engineering, Chinese Academy of Sciences, Sanya, China Deep-sea hydrothermal vent environments are characterized by high hydrostatic pressure and sharp temperature and chemical gradients. Horizontal gene transfer is Edited by: thought to play an important role in the microbial adaptation to such an extreme Philippe M. Oger, UMR CNRS 5240 Institut National environment. In this study, a 21.4-kb DNA fragment was identified as a genomic des Sciences Appliquées, France island, designated PYG1, in the genomic sequence of the piezophilic hyperthermophile Reviewed by: Pyrococcus yayanosii. According to the sequence alignment and functional annotation, Federico Lauro, University of New South Wales, the genes in PYG1 could tentatively be divided into five modules, with functions related Australia to mobility, DNA repair, metabolic processes and the toxin-antitoxin system. Integrase Amy Michele Grunden, can mediate the site-specific integration and excision of PYG1 in the chromosome of North Carolina State University, USA R Anaïs Cario, P. yayanosii A1. Gene replacement of PYG1 with a Sim cassette was successful. The Rensselaer Polytechnic Institute, USA growth of the mutant strain 1PYG1 was compared with its parent strain P. yayanosii *Correspondence: A2 under various stress conditions, including different pH, salinity, temperature, and Jun Xu [email protected] hydrostatic pressure. The 1PYG1 mutant strain showed reduced growth when grown at 100◦C, while the biomass of 1PYG1 increased significantly when cultured at Specialty section: 80 MPa. Differential expression of the genes in module III of PYG1 was observed This article was submitted to Extreme Microbiology, under different temperature and pressure conditions. This study demonstrates the first a section of the journal example of an archaeal integrative genomic island that could affect the adaptation of Frontiers in Microbiology the hyperthermophilic piezophile P. yayanosii to high temperature and high hydrostatic Received: 31 August 2016 pressure. Accepted: 16 November 2016 Published: 29 November 2016 Keywords: deep-sea, piezophilic hyperthermophile, Pyrococcus, genomic island, integrative element, adaptation Citation: Li Z, Li X, Xiao X and Xu J (2016) An Integrative Genomic Island Affects the INTRODUCTION Adaptations of the Piezophilic Hyperthermophilic Archaeon Deep-sea hydrothermal vent environments are characterized by high hydrostatic pressure (HHP) Pyrococcus yayanosii to High Temperature and High Hydrostatic and sharp temperature and chemical gradients (Reysenbach et al., 2006; Oger and Jebbar, 2010). Pressure. Front. Microbiol. 7:1927. The microorganisms dwelling here are expected to show strong high temperature adaptation doi: 10.3389/fmicb.2016.01927 (Jebbar et al., 2015). Mobile genetic elements, such as plasmids, bacteriophages, transposons, Frontiers in Microbiology | www.frontiersin.org 1 November 2016 | Volume 7 | Article 1927 fmicb-07-01927 November 25, 2016 Time: 18:18 # 2 Li et al. Genomic Island Affects Pyrococcus Adaptation integrons, conjugative transposons, integrative conjugative been identified among the eight Pyrococcus genomes (White elements (ICEs), and genomic islands (GIs), are important and et al., 2008), suggesting that maintenance of the microbial essential components of the marine biosphere that promote phenotypic diversity by extensive genome rearrangements marine microbial diversification (Sobecky and Hazen, 2009). and HGT help to respond to rapidly changing environmental Horizontal gene transfer (HGT) of mobile genetic elements is conditions. assumed to play an important role in the microbial adaptation to Pyrococcus yayanosii CH1 is the first example of the strictly extreme environments (van Wolferen et al., 2013). piezophilic hyperthermophilic archaeon isolated from the mid- Many of the accessory genes acquired by HGT form syntenic Atlantic Ridge hydrothermal vents (4,100-m depth; Zeng et al., blocks recognized as GIs (Juhas et al., 2009). These gene 2009; Birrien et al., 2011). A complete genome sequence of a 1.7- fragments are often inserted into tRNA gene loci, which act as Mb circular chromosomal DNA molecule of the model strain integration sites for foreign DNA, mainly prophages, and are P. yayanosii CH1 was announced (Jun et al., 2011), and a gene flanked by direct repeats (DRs) that consist of a few to more disruption system has been developed (Li et al., 2015). In the than a hundred nucleotides. Many GIs can spontaneously excise present study, a typical GI (PYG1) in the P. yayanosii genome was from and integrate into the chromosome, while some of them identified and genetically characterized. The mobility of PYG1 can lose this mobility. Integrases, transposases, ISs, and other was confirmed, and an artificial GI was constructed to investigate mobility genes encoded in GIs can be involved in the integration, the integration process. Moreover, growth differences between mobility, deletion, and rearrangement of GIs (Darmon and the parent strain and the PYG1 deletion mutant strain under high Leach, 2014). temperature and HHP suggested that this archaeal integrative GI The GC content and the codon usage of GIs are generally plays an important role in the environmental adaptation of this different from the other regions of the chromosome. GIs species. are typically recognized as discrete DNA segments between closely related strains, and these elements might contribute to the diversification and adaptation of microorganisms, MATERIALS AND METHODS thereby significantly impacting genome plasticity and evolution (Polz et al., 2013). For instance, seven novel cell Strains and Plasmids, Media and Growth wall-associated GIs delineated two major clades within Conditions the halophilic archaeon Haloquadratum walsbyi genome, The plasmids and strains used in the present study are listed in and this type of variation probably reflects a number of Table 1. P. yayanosii A1, a facultative piezophilic derivative strain mechanisms that minimize the infection rate of viruses of P. yayanosii CH1, was cultivated in 100 ml serum bottles under (Martin-Cuadrado et al., 2015). The transcriptome of the anaerobic conditions at 95◦C and 0.1 MPa in 30 ml of TRM (Zeng piezophile Photobacterium profundum SS9 grown under et al., 2009; Li et al., 2015) containing 3.3 g PIPES disodium salt, ◦ different pressure (28 MPa vs. 45 MPa) and temperature (4 C 30 g NaCl, 5 g MgCl2 6H2O, 0.7 g KCl, 0.5 g (NH4)2SO4, 1 ml ◦ vs 16 C) conditions was analyzed, and the results showed KH2PO4 5%, 1 ml K2HPO4 5%, 1 ml CaCl2 2H2O 2%, 0.05 g that there were differentially expressed genes that belonged NaBr, 0.01 g SrCl2 6H2O, 1 ml Na2WO4 10 mM, 1 ml FeCl3 to three GIs (Chr1.8, Chr2.3, and Chr2.5) in the SS9 genome; 25 mM, 1 g yeast extract, 4 g tryptone, and 1 mg resazurin. After these genes are absent in both the pressure-sensitive strain transformation, the strains were selected on TRM supplemented 3TCK and the pressure-adapted strain DSJ4 (Campanaro et al., with 10 mM simvastatin (Sigma). Gelrite (1.5% w/v) was added to 2005). In addition, genes that are responsible for the defense solidify the medium. The medium pH was adjusted from 5.8 to function, including the toxin-antitoxin system, restriction- 8.2 by adding 1 M HCl or 1 M NaOH. The salinity of the medium modification system, phage abortive infection system, and was adjusted by adding different amounts of NaCl. The growth CRISPR/Cas system, were frequently identified in GIs in various was monitored by cell counting using a Thomas chamber and bacteria and archaea (Dobrindt et al., 2004; Makarova et al., light microscopy at a magnification of ×40 (Zeng et al., 2009). 2011). The Escherichia coli strain DH5a was used for general DNA Thermococcales are widely distributed in geothermal manipulation, and the E. coli was cultivated in Luria-Bertani (LB) environments, including hot springs, volcanoes, and deep-sea medium at 37◦C. hydrothermal vents (Bertoldo and Antranikian, 2006; Stetter, 2006; Ferrera and Reysenbach, 2007). The order Thermococcales HHP Culturing Experiments is represented by three genera, Thermococcus, Pyrococcus, All manipulations before the pressurized culturing experiments and Paleococcus, which are obligate anaerobic heterotrophic were performed anaerobically inside an anaerobic glove box (Coy hyperthermophiles (Bertoldo and Antranikian, 2006; Ferrera Lab). Cultivation of P. yayanosii was performed using a custom- and Reysenbach, 2007). Genetic elements with the characteristics built high pressure/high temperature incubation system similar of GIs have also been identified in Thermococcales. Four virus- to that reported by Zeng et al.(2009). A 10-ml plastic syringe was like regions (TKV1 through TKV4) have been found in the used as the container of liquid medium. After inoculation, the genome of T. kodakarensis (Fukui et al., 2005), and the genes needle head of the syringe was